My PhD research is the identification and characterization of genes for inherited bone marrow failure and myelodysplastic syndromes (BMF/MDS). Since my previous F30 submission, I have identified two candidate genes for these conditions by genomic analysis of severely affected families. The aims of this new proposal are to test each gene as the possible cause of inherited BMF/MDS in its host family, then to evaluate the possible role of each gene in hematopoiesis. In Aim 1, I will test whether a missense mutation in GALE (UDP-galactose-4-epimerase) may be responsible for severe thrombocytopenia, febrile neutropenia, and anemia in a large consanguineous kindred. In Aim 1a, I will screen for other mutations in GALE by fully sequencing the gene in DNA from unrelated patients from the BMF/MDS repositories. In Aim 1b, I will compare enzymatic activities of wildtype and mutant GALE by HPLC. I will also compare the crystal structures of wildtype and mutant GALE in complex with NADH and nucleotide sugars. In Aim 1c, I will suppress GALE in primary human CD34+ hematopoietic stem cells and measure cell proliferation, differentiation, and survival. I will also explore ER stress and other effects that may be mechanisms by which loss of GALE impairs hematopoietic development. In Aim 2, I will test whether a missense mutation in transcription factor TFDP2 (E2F Dimerization Partner-2) may be responsible for severe thrombocytopenia, anemia, and craniosynostosis in affected siblings in a consanguineous family. In Aim 2a, I will screen for other mutations in TFDP2 in DNA from patients from the BMF/MDS repositories and, independently, in patients with craniosynostosis. In Aim 2b, I will compare mutant and wildtype TFDP2 with respect to binding affinity to E2F partners and will compare effects of wildtype and mutant E2F/TFDP2 complexes on transcription activity of target genes. In Aim 2c, I will test the effects of suppression of TFDP2 on CD34+ cells, and test for defects in cell cycle transition and in DNA replication by comparing endoreduplication levels and BrdU incorporation. Elucidating roles for GALE and TFDP2 in hematopoiesis will add important knowledge to the field. Studying the role of GALE can help explain how changes in glycosylation affect hematopoietic proliferation and differentiation. E2F pathways are well-known in cell cycle regulation, but less so in development. Studying mutation in TFDP2 will improve our understanding of the role of E2F/TFDP transcription factor complexes in hematopoiesis and human development.